Inactivation of the panE Gene in Lactococcus lactis Enhances Formation of Cheese Aroma Compounds

Abstract

bHydroxyacid dehydrogenases limit the conversion of ␣-keto acids into aroma compounds. Here we report that inactivation of the panE gene, encoding the ␣-hydroxyacid dehydrogenase activity in Lactococcus lactis, enhanced the formation of 3-methylbutanal and 3-methylbutanol. L. lactis IFPL953⌬panE was an efficient strain producing volatile compounds related to cheese aroma. Flavor formation in dairy products is directly related to lactic acid bacteria (LAB), which are equipped with a large number of key intracellular enzymes responsible for the formation of volatile compounds from amino acid catabolism (1, 2). In Lactococcus lactis, amino acid degradation is initiated by a transamination reaction, catalyzed mostly by AraT and BcaT aminotransferases, that produces the corresponding ␣-keto acid (3, 4). The ␣-keto acids are subsequently degraded either into aldehydes, which are further converted into alcohols, carboxylic acids, and esters, or into ␣-hydroxy acids, which are not aroma compounds (1). Two key enzymes are involved in these reactions, an ␣-ketoacid decarboxylase (KivD) responsible for the transformation of the ␣-keto acids into aldehydes (5) and an ␣-hydroxyacid dehydrogenase (PanE) responsible for the conversion of the ␣-keto acids into the nonaromatic ␣-hydroxy acids (6). Hydroxyacid dehydrogenases (HA-DHs) negatively affect aroma production by competing with decarboxylases for ␣-keto acids (7). In order to optimize the degradation of ␣-keto acids into volatile compounds, we have inactivated the panE gene in L. lactis IFPL953. The strain was isolated from raw milk cheese and was selected because it possesses glutamate dehydrogenase (GDH) activity, to produce the ␣-ketoglutarate required for AraT and BcaT transamination reactions, and KivD activity, which is needed for the transformation of ␣-keto acids into aldehydes (8).The HA-DH activity in L. lactis IFPL953 was inactivated by double-crossover mutation using the thermosensitive pG ϩ host9 vector (9) containing a 408-bp deletion in the panE gene. All primers used for PCR amplification and DNA sequencing during the inactivation of the panE gene are shown in Table 1. The plasmid pGϩ9::⌬panE was produced in Escherichia coli TIL206 (10), selected for erythromycin resistance (150 g/ml), and transformed into L. lactis IFPL953 by electroporation (11). L. lactis IFPL953⌬panE was achieved by chromosome integration of pGϩ9::⌬panE at 37°C and excision at 28°C. The mutation was verified by sequencing the 1,645-bp PCR product ⌬panE (Table 1). L. lactis IFPL953⌬panE and the wild-type strain were analyzed for HA-DH activity using ␣-ketoisocaproic acid (KIC) as the substrate (6), showing L. lactis IFPL953 values of 5.78 U/mg (1 U is defined as the quantity of enzyme that oxidizes 1 mol NADH per minute at 37°C), whereas the mutant strain had lost the HA-DH activity. The effect of the mutation on L. lactis IFPL953⌬panE growth was tested by comparing the growth to that of L. lactis IFPL953 grown in a buffered chemically defined medium (CDM) (12) for 24 h (Fig. 1A) and i...

Expression of amino acid converting enzymes and production of volatile compounds by Lactococcus lactis IFPL953

“…Overall, the lack of the α-hydroxyacid dehydrogenase (HA-DH or PanE) activity did not change the L. lactis IFPL953 growth profile under BCAA starvation conditions. Similar growth behaviour was previously observed when both strains were grown in milk (Gómez de Cadiñanos et al, 2013).…”

“…2). The inactivation of the panE gene, which limits the formation of α-hydroxy acids (Gómez de Cadiñanos et al, 2013), was related with higher expression of the kivD and ytjE genes and decreased expression of araT and bcaT (Fig. 2).…”

“…Further catabolism of these α-keto acids is catalyzed by an α-ketoacid decarboxylase (KivD) responsible for their conversion into aldehydes (De la Plaza, Fernández de Palencia, Peláez, & Requena, 2004;. Hydroxy acid dehydrogenases (HA-DHs) negatively affect aroma production by competing with decarboxylases for α-keto acids to convert them into the non-aromatic α-hydroxy acids (Broadbent et al, 2004;Chambellon et al, 2009;Gómez de Cadiñanos et al, 2013).…”

“…L. lactis IFPL953 is a wild strain isolated from raw milk cheese characterized by a wide enzymatic background for aroma production, including AraT and BcaT activities, the GDH activity to produce αketoglutarate required for transamination reactions, and the KivD and C-S lyase (YtjE) activities. In addition, we have inactivated its HA-DH activity (encoded by the panE gene) to generate L. lactis IFPL953ΔpanE in order to optimize the degradation of αketo acids into volatile compounds (Gómez de Cadiñanos et al, 2013). The objective of the present work has been to investigate the effect of BCAA content in the growth medium on the expression of genes codifying AACE and the formation of volatile compounds by L. lactis IFPL953 and the mutant L. lactis IFPL953ΔpanE.…”

Dietary Probiotic Effect of Lactococcus lactis WFLU12 on Low-Molecular-Weight Metabolites and Growth of Olive Flounder (Paralichythys olivaceus)

“…It has been revealed that HICA can increase weight and muscle mass in chickens and rats (Boebel and Baker, 1982 ). In the present study, genes encoding enzyme 3-hydroxyisobutyrate dehydrogenase for conversion of 3HBA to acetoacetic acid and L-2-hydroxyisocaproate dehydrogenase (HicDH) for HICA production (de Cadiñanos de et al, 2013 ) were found in our probiotic genome (Table 2 ). However, functions of these metabolites in fish are not yet fully understood.…”